Pycnogenol^® Mitigates Oxidative Stress and Improves Skin Defenses Against Environmental Pollutants: An Ex-Vivo Human Skin Explant Study

Abstract

Oxidative stress is a major factor in skin aging and various skin pathologies. Environmental pollutants exacerbate this stress by generating reactive oxygen species (ROS), disrupting the skin’s redox balance. Pycnogenol^®, a French maritime pine bark, extract is standardized to contain 70 ± 5% procyanidins and known to mitigate oxidative damage and inflammation. This study aims to evaluate the potential antipollution and antioxidant effects of Pycnogenol^® on skin. Ex vivo human skin explants were treated with varying concentrations of Pycnogenol^® (0.5%, 1%, and 2%) and then exposed to a mixture of pollutants. The expression of stress markers Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) and AHR (Aryl Hydrocarbon Receptor) were evaluated using immunostaining. Lipid peroxidation levels were measured by quantifying malondialdehyde (MDA) concentrations. The extract significantly decreased Nrf2 expression by 40% (p = 0.003) and 23% (p = 0.048) with a dose of 2% and 1%, respectively. After pollutant exposure, Pycnogenol^® (0.5%, 1%, and 2%) reduced Nrf2 over-expression in a dose–response manner by 29% (p = 0.03), 58% (p = 0.004) and 64% (p = 0.002) respectively. Pycnogenol^® at 0.5%, 1%, and 2% significantly reduced AHR over-expression by 61% (p < 0.0001), 76% (p < 0.0001) and 85% (p < 0.0001), respectively. Pycnogenol^® (1%, and 2%) decreased MDA levels following pollutant exposure by 17% (p = 0.06) and 25% (p = 0.01) respectively. In a dose-dependent manner, Pycnogenol^® exhibited a strong protective effect against pollution, significantly reducing pollutant-induced basal oxidative stress (MDA) and over-expression of Nrf2 and AHR, key factors in oxidative stress and detoxification. Pycnogenol^® also increased AHR expression in the absence of pollutants, which may reflect an adaptive cellular response.

1. Introduction

Oxidative stress of the skin, characterized by an imbalance between reactive oxygen species (ROS) and the skin’s antioxidant defenses [1], is a significant factor in skin aging [2,3], inflammation [4], and various dermatological conditions [5]. This imbalance occurs when there is an overproduction of ROS, including free radicals such as superoxide anions (O₂•−) and hydroxyl radicals (•OH), as well as non-radical species like hydrogen peroxide (H₂O₂) [6]. These reactive molecules can damage cellular macromolecules such as lipids, proteins, and DNA, resulting in functional impairments and cellular apoptosis [1,7].

Environmental pollution, stemming from sources such as vehicle emissions, industrial activities, and tobacco smoke, exacerbates oxidative stress in the skin [8]. Pollutants such as particulate matter (PM), heavy metals like lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As), or nitrogen oxides (NO_x), and ozone (O₃) can penetrate or affect the skin barrier and generate ROS through several mechanisms [9,10]. These include direct generation of ROS [11], triggering inflammatory responses in skin cells [12], and interaction with cellular components that form additional ROS [13]. Such interactions disrupt the skin’s redox balance, contributing to premature aging [14], inflammation, and increased susceptibility to skin diseases [8,9].

In response to atmospheric pollution, the skin activates a series of defense mechanisms targeting oxidative stress, lipid peroxidation, and xenobiotic response. This response includes the transcription factor Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2), the oxidative stress marker Malondialdehyde (MDA), and the Aryl Hydrocarbon Receptor (AHR).

Nrf2 can be seen as a primary cellular response to oxidative stress and this transcription factor plays a major role in protecting human skin keratinocytes from oxidative damage. In more detail, Nrf2 regulates the expression of antioxidant and detoxification enzymes [15]. Normally, Nrf2 is kept in the cytoplasm by its inhibitor KEAP1 (Kelch-like ECH-associated protein 1). In response to oxidative stress, Nrf2 dissociates from KEAP1, and activates genes with antioxidant response elements (AREs) [16,17] Nrf2 activation also mitigates inflammation and supports skin repair processes. However, chronic exposure to pollutants can overwhelm Nrf2, leading to exacerbated skin damage [18].

MDA is a key by-product of lipid peroxidation, a process in which ROS attack polyunsaturated fatty acids in cell membranes [19]. Elevated MDA levels therefore indicate oxidative stress and cellular damage, as MDA can form adducts with proteins and DNA, further disrupting cellular function [20]. Increased MDA levels are associated with various skin conditions, including aging, photoaging, and inflammatory disorders [21,22]. Measuring MDA in skin biopsies or culture media provides insight into oxidative damage and serves as a valuable biomarker for evaluating antioxidant therapies. Environmental pollutants such as heavy metals significantly increase MDA levels in the skin by accelerating lipid peroxidation, leading to higher MDA concentrations and contributing to oxidative stress-related skin damage [23].

AHR is a ligand-activated transcription factor involved in regulating xenobiotic metabolism and responding to environmental toxins [24]. It is involved in the activation of cytochrome family genes and detoxification enzymes. AHR is activated following exposure to several compounds, including polycyclic aromatic hydrocarbons (PAH) and ozone. When bound to ligands such as PAH, AHR translocates to the nucleus and activates genes involved in detoxification and inflammatory responses. However, AHR is a double-edged sword in skin health: it aids in detoxifying harmful substances and protecting against environmental stressors, but excessive or prolonged AHR activation can lead to inflammation and skin damage [25]. For instance, AHR activation by pollutants can induce the expression of pro-inflammatory cytokines and disrupt the skin barrier, contributing to inflammatory skin conditions and increased oxidative stress [26]. Pollutants such as PAH, found in cigarette smoke and vehicle or industrial emissions, are potent AHR ligands, and their interaction with AHR can exacerbate oxidative stress and inflammation in the skin [26,27].

In this context, Pycnogenol^®, a patented procyanidin-rich extract from French maritime pine bark, has emerged as a key mitigator of oxidative damage and inflammation [28]. Pycnogenol^® is also rich in other bioflavonoids, including catechin, epicatechin, taxifolin, and phenolic acids such as ferulic and caffeic acids [29]. Its effectiveness stems from its ability to scavenge reactive oxygen and nitrogen species, offering substantial antioxidant and anti-inflammatory benefits [28].

Among the various techniques suitable to model anti-pollution effects involving the surface of the skin, the Pollubox^® system using ex vivo human explants [30] stands out, as it allows the measurement of relevant biological markers directly in the cells of human skin, after topical product application. This is a departure from convoluted extrapolations from animal or in vitro studies typically used to assess the properties of cosmetic ingredients. As even skin morphology, structure and metabolism are preserved with this set-up, this model provides a relevant context for studying the effects of topically applied ingredients.

With the Pollubox system, the Nrf2, MDA, and AHR parameters can be analyzed individually to characterize the overall skin response to oxidative stress and environmental insults.

Nrf2-mediated gene regulation is a central cellular defense mechanism that orchestrates the expression of antioxidant enzymes and cytoprotective proteins, thereby mitigating oxidative damage caused by environmental stressors. MDA (malondialdehyde) is a well-established biomarker of lipid peroxidation and provides a direct quantitative measure of oxidative stress experienced by the tissue. AHR (aryl hydrocarbon receptor), in contrast, plays a dual and context-dependent role: it is critical for sensing and detoxifying environmental pollutants, yet its excessive or prolonged activation can trigger pro-inflammatory signaling and exacerbate oxidative damage. For these reasons, this triad represents a rational choice for evaluating the antioxidant activity of a molecule in ex vivo studies on human skin explants, as it encompasses both protective mechanisms, deleterious effects, and indicators of oxidative stress.

In the last decade, the negative effects of environmental pollution on the skin have been evidenced in several studies [9,12,31,32], resulting in increased consumer awareness and demand for pollution-protecting solutions to cosmetic products. In this context, the cosmetic industry welcomes innovative active ingredients that leverage different mechanisms of action to protect the skin from the harmful effects of environmental pollution [33,34,35,36,37,38,39]. Oral intake of Pycnogenol^® has shown to have beneficial effects on elasticity and skin moisture in urban outdoor workers, subjected to high pollution levels [33]. However, after oral ingestion, the extract is metabolized, and its action differs from direct application on the skin [29]. Studying Pycnogenol^® on human skin explants therefore provides new evidence of its ability to protect the skin from pollution-related oxidative damage through direct local effects.

The objective of this study is therefore to evaluate the anti-pollution activity of topical Pycnogenol^® on the skin, by assessing the most relevant oxidative stress pathways and markers.

2. Materials and Methods

2.1. Skin Preparation

Thirty-five human abdominoplasty skin explants were utilized for this study. Explants were obtained from a 58-year-old Caucasian female donor. The studies were conducted in accordance with the Declaration of Helsinki and after the patients had given informed consent. Each explant had an average diameter of 12 mm (±1 mm) and a surface of 1 cm² and was placed in BEM culture medium (BIO-EC’s Explants Medium, Eurofins BIO-EC, Longjumeau, France) to survive and maintained at 37 °C in a humid environment with 5% CO₂. Explants (n = 4 per treatment) were then distributed according to the treatment and sampling schedule shown in

Table 1. Overview of the experimental groups and treatments used in this study. Further details of concentration, stress conditions, and sampling times are provided in the text.

Batch Identity	Treatment	Number of Explants	Sampling Time
Control	Untreated skin	4	day 5
P1	Pycnogenol® at 0.5%	4
P2	Pycnogenol® at 1%	4
P3	Pycnogenol® at 2%	4
Stress	Pollutant mixture	4
P1 + Stress	Pollutant mixture + Pycnogenol® at 0.5%	4
P2 + Stress	Pollutant mixture + Pycnogenol® at 1%	4
P3 + Stress	Pycnogenol® at 2% + Pollutant mixture	4

and

Table 2. Schematic representation of the experimental protocol used in the present work. Human skin samples were treated with Pycnogenol^® at different concentrations (0.5%, 1%, and 2%) with or without pollutant-induced stress. Treatments and sampling points were distributed over a five-day period as indicated by the symbols: ▲ Pycnogenol^® treatment, ■ pollutant mixture treatment, and ▼ sampling time.

Batch	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5
Untreated skin						▼
Pycnogenol® at 0.5%	▲			▲		▼
Pycnogenol® at 1%	▲			▲		▼
Pycnogenol® at 2%	▲			▲		▼
Stress condition					■	▼
Pycnogenol® at 0.5% + stress	▲			▲	▲ ■	▼
Pycnogenol® at 1% + stress	▲			▲	▲ ■	▼
Pycnogenol® at 2% + stress	▲			▲	▲ ■	▼

. All experimental procedures, including the preparation of human skin explants, the application of treatments, the renewal of culture medium, and the preparation of test products, were carried out under strictly controlled sterile conditions. Throughout the entire workflow, aseptic techniques were rigorously applied to prevent contamination and to ensure the reliability and reproducibility of the experimental outcomes.

2.2. Test Products & Schedule

(i) Test Products—Pycnogenol^® (Horphag Research) was dissolved in sterile distilled water at a concentration of 0.5%, 1% and 2% w/w, resulting in complete solubility of the test product at all used concentrations. Pycnogenol^® is a proprietary product made exclusively from French maritime pine (Pinus pinaster subsp. atlantica) bark extract. The trees are cultivated as a monoculture exclusively in a narrow area in southwestern France (Landes de Gascogne). The forest is the largest found in Europe, with 2.5 million acres, the majority of which is a national park. The entire process is completely sustainable as cut trees are replaced by seedlings, which is controlled by the French Government. The multilayered thick outer bark is harvested from 30-year-old cultivated trees grown for timber. Timber production generates far more bark as a byproduct than what is required for extraction of Pycnogenol^®. Research suggests significant antioxidant activity for Pycnogenol^®, based primarily on its procyanidin content, standardized to 70% +/− 5% [29].

(ii) Pollution Treatment—Pollutant exposure was performed using two approaches: a commercially available ready-to-use solution and individual compounds, including benzene, toluene, xylene, and diesel particles, which are well-known environmental contaminants frequently encountered in urban areas. In preliminary experiments conducted prior to the present study, maximum concentrations of these pollutants were established that do not compromise cellular or tissue viability while still modulating key markers of oxidative stress. Translating these concentrations to real-life exposure is challenging, as pollution levels vary significantly between cities and across seasons, and using actual environmental pollutant concentrations rarely produces measurable effects on skin physiology ex vivo. The aim of the present study was therefore to qualitatively reproduce environmental pollution and develop an “accelerated biological model” to investigate the impact of environmental pollutants on the skin. The pollutants mixture used was composed by a standardized heavy metals solution (Merck, Darmstadt, Germany), supplemented with polycyclic aromatic hydrocarbons benzene (Fluka (Sigma-Aldrich, Merck), Darmstadt, Germany) and xylene (Fluka (Sigma-Aldrich, Merck), Darmstadt, Germany), toluene (Sigma-Aldrich (Merck), Darmstadt, Germany), and diesel particulate matter (National Institute of Standards and Technology, Gaithersburg, MD, USA) (

Table 3. Composition of the pollutant mixture used in the study.

Identification	Provider	Reference	Final Concentration
Solution ICP multi-element standard V	Merck	1.10714.0500	See Table 4
Benzene	Fluka	12550	0.001%
Xylene	Fluka	95673	0.001%
Toluene	Sigma	34866	0.001%
Diesel particulate matter	NIST	NIST1650B	0.01%

and

Table 4. Concentrations of individual elements and heavy metals, included in the pollutant mixture. Values are expressed in mg/mL.

Elements	Concentration (mg/mL)
Al	0.01
As	0.01
B	0.001
Ba	0.001
Be	0.0005
Ca	0.005
Cd	0.001
Cr	0.001
Cu	0.001
Fe	0.001
Hg	0.0025
K	0.0495
Li	0.001
Mg	0.0005
Mn	0.0005
Na	0.01
Ni	0.0025
P	0.005
Pb	0.01
Sc	0.0005
Se	0.01
Sr	0.0005
Te	0.01
Ti	0.001
Y	0.0005
Zn	0.001

).

(iii) Product Application—On day 0 (D0), D3 and D4 (4 h before pollutant exposure), Pycnogenol^® at 0.5%, 1% and 2% was topically applied, 2 μL per explant (2 µL/cm² a dose that is commonly used to ensure the reproducibility of the results) and spread using a small spatula (Sigma-Aldrich (Merck), Darmstadt, Germany). The untreated explants did not receive any treatment except the refreshing of the medium every two days.

(iv) Pollution Exposure & Sampling—On day 4, four hours after product application, explants were placed in a PolluBox^® system [30], containing 900 μL of HBSS (Hank’s Balanced Salt Solution) (Gibco, Thermo Fisher Scientific; Waltham, MA, USA. Ref. 14025-050) per well. They were then exposed to a mixture of polycyclic aromatic hydrocarbons, heavy metals, and particulate matter, supplemented with 0.9% NaCl (Sigma-Aldrich (Merck), Darmstadt, Germany, ref. S304-500G) (150 μL of 0.9% NaCl per ml of pollutant solution) for 1.5 h, by spraying, using 1.5 mL of the mixture according to the treatment and sampling schedule shown in Table 1 and Table 2. The time of 1.5 h was determined in previous experiments to be required for the nebulized pollutant-containing solution to be fully deposited on the surface of the skin explants. Untreated skin explants and explants treated with only Pycnogenol^® at the different concentrations (P1–P3) were maintained in 1 mL of HBSS during this period. At the end of the pollution exposure treatment, all explants were removed and returned to 2 mL fresh BEM medium.

2.3. Histological Processing

After fixation for 24 h in buffered formalin, explants were dehydrated and impregnated in paraffin using a HistoCore PEARL dehydration automat (Leica Biosystems, Wetzlar, Germany) and then embedded using a Leica EG 1160 embedding station (Leica Biosystems, Wetzlar, Germany). 5 μm-thick sections were cut using a Leica RM 2125 Minot-type microtome (Leica Biosystems, Wetzlar, Germany) and then mounted on Superfrost^® histological glass slides (Epredia, Breda, Netherlands, ref. J1800AMNZ). Frozen samples were cut into 7 μm-thick sections using a Leica CM 3050 cryostat (Leica Biosystems, Wetzlar, Germany). Sections were then mounted on Superfrost^® silanised glass slides. Microscopic observations were recorded using an Olympus BX43 microscope (Evident, formerly Olympus, Tokyo, Japan). Pictures were digitized with a numeric DP72 Olympus camera (Evident, Tokyo, Japan) with cellD storing software (version 3.4, Evident, Tokyo, Japan).

2.4. Microscopy

Cell viability of the epidermal and dermal structures in all skin explants, was assessed by microscopical observation of formalin-fixed paraffin-embedded (FFPE) skin sections after Masson’s trichrome staining, Goldner variant [40]. Staining was assessed by microscopic observation.

For Nrf2 assessment, FFPE-skin sections were incubated with a monoclonal anti-Nrf2 antibody (Abcam, Cambridge, UK, ref. ab76026, clone EP1809Y) diluted at 1:200 in PBS (phosphate-buffered saline) (Sigma Aldrich, (Merck), Darmstadt, Germany, ref. P3813-10PAK) containing 0.3% BSA (bovine serum albumin) (Sigma Aldrich (Merck), Darmstadt, Germany, ref. A9085-25G) and 0.05% Tween 20 (Sigma Aldrich, (Merck), Darmstadt, Germany, ref. P1379-25ML). The sections were incubated for 1 h at room temperature with the Vectastain Kit Vector amplifier system (avidin/biotin) (Vector Laboratories, Burlingame, CA, USA, ref. SK-7200), and the staining was visualized with VIP, a peroxidase substrate (Vector Laboratories, Burlingame, CA, USA, ref. SK-4600). The entire immunostaining process was carried out using an automated slide processing system (Autostainer, DAKO, Glostrup, Denmark) and evaluated by microscopic observation.

AHR immunostaining and visualisation was performed on FFPE skin sections using a monoclonal anti-AHR antibody (Thermo Scientific, Waltham, MA, USA, ref. MA1-514, clone RPT1) diluted at 1:100 in PBS with 0.3% BSA and 0.05% Tween 20. The sections were incubated for 1 h at room temperature with the Vectastain Kit Vector amplifier system (avidin/biotin), and the staining was visualized with VIP, a peroxidase substrate (Vector Laboratories, Burlingame, CA, USA, ref. SK-4600). The immunostaining process was carried out using an automated slide processing system (Autostainer, DAKO, Glostrup, Denmark) and assessed by microscopic observation.

2.5. Lipid Peroxidation Assay (MDA)

The malondialdehyde (MDA) assay was conducted using an enhanced version of the TBARS (thiobarbituric acid reactive substances) assay. MDA was measured in HBSS medium by adding TBARs solution, containing thiobarbituric acid (Sigma, (Merck), Darmstadt, Germany, ref. T5500-25G), hydrochloric acid (VWR Chemicals, Leuven, Belgium. Ref. 30024.290), and trichloroacetic acid (Sigma-Aldrich, (Merck), Darmstadt, Germany, ref. T4885) and incubating the mixture in a water bath at 80 °C for 15 min. To improve assay specificity, MDA was extracted using liquid/liquid extraction with butanol (Sigma Aldrich, (Merck), Darmstadt, Germany, ref. 20808.462), as many substances (e.g., glucose) that are not related to lipo-peroxidation can react with thiobarbituric acid. The MDA in the butanol extract was quantified using spectrofluorimetry (excitation at 515 nm, emission at 550 nm) with a Tecan Infinite M200 Pro micro-plate reader (Tecan, Männedorf, Switzerland). This assay measured the MDA concentration in the culture medium on day 5, and the MDA concentration was expressed in nmol/L (n = 3–4). Because the sample size was limited (n = 3–4), the statistical power of the analyses was inherently low, and the results should therefore be interpreted with caution.

Although the use of butanol extraction improves selectivity, the TBARS method remains an indirect measure of lipid peroxidation and may be influenced by other aldehyde byproducts. Therefore, the results should be interpreted as an overall indicator of oxidative stress rather than as a specific quantification of MDA alone.

2.6. Image Analysis

All image analyses were performed on all the images on each batch, according to cellD software (Evident, Tokyo, Japan) methods. For each batch of skin explants, the percentage of the region of interest covered by the staining (i.e., stained surface percentage) was then determined, and the stained surface percentage for each treatment was then compared to the untreated condition. A region of interest (ROI) was first defined for each image (e.g., viable epidermis). Pixel detection settings were then established by adjusting the hue, saturation, and intensity (HSV threshold) to isolate the specific staining signal. These parameters were subsequently applied uniformly to all images.

2.7. Statistical Analysis

The Shapiro–Wilk (https://www.statskingdom.com) test was first applied to assess the normality of the data. When the data were normally distributed, an unpaired t-test was used for statistical analysis. When normality was not confirmed, the Mann–Whitney test was applied (https://www.socscistatistics.com). The significance level for this study was established as follows: if p < 0.05 (*), this indicated a 95% probability of significant difference; and if p < 0.01 (**), this indicated a 99% probability of significant difference. Raw data of each analysis are reported in Supplementary Tables S1–S3.

3. Results

The treatment with Pycnogenol^® at 0.5%, 1% and 2% did not induce any change in cell viability in both normal condition and upon pollutant exposure (Figure 1) after 5 days of treatment. Cell viability was also assessed on day 0 on freshly prepared human skin explants, to confirm that the samples exhibited adequate initial viability.

When comparing the effects of Pycnogenol^® on Nrf2 immunostaining (Figure 2) to untreated skin under normal condition, we observed a significant decrease of 40% (p < 0.003), with the dose at 2% and a significant decrease of 23% (p < 0.048) with the dose of 1%, while a non-significant increase of 12% is observed with the dose at 0.5%.

Pollutant exposure resulted in a significant increase in Nrf2 immunostaining by 60% (p < 0.008) in control skin explants. When evaluating the effects of Pycnogenol^® on Nrf2 activation compared to pollutants-exposed skin, we observed a dose-dependent decrease (Figure 1). A significant decrease of 29% (p < 0.029), 58% (p < 0.0004) and 64% (p < 0.0002) was observed following Pycnogenol^® application at 0.5, 1% and 2% respectively.

The surface percentage positive to AHR immunostaining in the living epidermis is shown in Figure 3. On day 5, in the blank batch, 20.6% ± 6.1 of the living epidermis surface was positive for AHR immunostaining suggesting a moderate expression of AHR. When comparing the effects of Pycnogenol^® application on AHR expression, with the untreated skin, Pycnogenol^® at 0.5% induced a non-significant decrease of 1%, Pycnogenol^® at 1% induced a significant increase of 58% (p < 0.016), and Pycnogenol^® at 2% induced a significant increase of 110% (p < 0.0001). Pollutant exposure resulted in a significant increase in AHR-activation of 198% (p < 0.0001) in control skin explants compared to untreated skin. When evaluating the effects of Pycnogenol^® on AHR expression upon pollutants exposure we observed a significant decrease of 61% (p < 0.0001) with Pycnogenol^® at 0.5%, a significant decrease of 76% (p < 0.001) with Pycnogenol^® at 1% and a significant decrease of 85% (p < 0.0001) with Pycnogenol^® at 2%.

MDA released in BEM culture media on day 5 (in nmol/L) is shown in Figure 4. On day 5, the mean concentration of MDA released in the BEM culture medium by untreated skin was 94.4 ± 2.3 nmol/L. When comparing the effects of Pycnogenol^® application on MDA concentration with untreated skin, Pycnogenol^® at 0.5% induced a non-significant decrease of 4%, Pycnogenol^® at 1% induced a non-significant decrease of 10% (p < 0.066), and Pycnogenol^® at 2% induced a significant decrease of 12% (p < 0.005). Pollutant exposure of control skin explants resulted in a significant increase of 14% (p < 0.089) in the concentration of MDA released in the culture medium compared to untreated skin. When evaluating the effects of Pycnogenol^® application on MDA concentration after pollutant exposure compared to pollutants-exposed control skin, Pycnogenol^® at 0.5% a non-significant decrease of 10%, Pycnogenol^® at 1% induced a non-significant decrease of 17% (p < 0.068), while Pycnogenol^® at 2% induced a significant decrease of 25% (p < 0.01).

4. Discussion

The present work demonstrates significant efficacy of Pycnogenol^® in providing a range of protective effects for the skin against pollution. These effects are shown to be dose-dependent.

Pycnogenol^® dose-dependently exhibited anti-pollution activity by reducing Nrf2 and AHR over-expression resulting from exposure to pollutants. AHR expression in the absence of pollutants was increased with 1 and 2% Pycnogenol^®, which may enhance xenobiotic detoxification by upregulating phase I and phase II metabolizing enzymes and strengthen antioxidant defenses through activation of pathways such as Nrf2, thereby helping to limit oxidative stress and cellular damage. Nrf2 expression was reduced dose-dependently as well, in the absence of pollutants, indicating a beneficial decrease in basal oxidative stress levels. Oxidative stress (MDA) induced by pollutants was reduced with 1 and 2% Pycnogenol^®.

Nrf2 can be seen as a primary response to oxidative stress and this transcription factor plays a major role in protecting human skin keratinocytes from oxidative damage, including that induced by UV-A radiation [17].

Ideally, an active compound or product should not modify or should slightly increase Nrf2 expression before pollutant exposure and reduce its over-expression after exposure. Reducing Nrf2 expression without pollutant exposure may be interpreted as an indication of a decrease in the basal oxidative stress level, while reducing Nrf2 over-expression after pollutant exposure reflects a decrease in the acute cellular damage and deteriorating effects of the pollutant as the need for a massive compensatory defense is lessened.

The observed reduction in Nrf2 expression after Pycnogenol^® treatment, both in response to environmental pollutants and in their absence, suggests a regulatory effect on the Nrf2 pathway that may help maintain redox balance and limit pollutant-driven cellular damage.

AHR is involved in activation of cytochrome family genes and detoxification enzymes and is activated following exposure to environmental pollutants such as PAH and ozone. Pycnogenol^® strongly repressed the over-expression of AHR induced by exposure to pollutants. Moreover, in the absence of pollutants, Pycnogenol^® increased AHR expression, implying that it promotes the skin aptitude to trigger a response against pollution-associated harmful effects.

MDA is a marker of lipid peroxidation, which indicates oxidative stress and cellular damage, and is associated with various skin conditions [21,22]. Application of a 2% Pycnogenol^® solution on the skin was found to be efficient at reducing lipid peroxidation significantly and thus decreasing oxidative stress in the skin and protecting it from pollution-induced damage.

Pycnogenol^® was shown to modulate the skin response to environmental insults for the 3 key parameters Nrf2, AHR and MDA.

While Nrf2, MDA, and AHR represent key pathways in the skin’s defense against atmospheric pollution, could further investigate additional components of the skin’s protective network. Inflammatory mediators such as NF-κB and cytokines including IL-6 and TNF-α are central to the regulation of cutaneous immune responses and inflammation triggered by environmental stressors. Markers of oxidative stress and lipid peroxidation, such as 4-HNE and protein carbonylation, provide insight into cumulative oxidative damage to cellular membranes and proteins. Detoxification enzymes, including CYP1A1, reflect the skin’s capacity to metabolize and neutralize xenobiotics. Evaluating these additional markers could offer a more comprehensive understanding of how the skin senses, responds to, and mitigates the damaging effects of pollutants, linking molecular signaling to functional outcomes such as barrier integrity, antioxidant defense, and inflammatory control.

The observed effects in the present study could be contextualized and enable us to better understand previous findings. In a previous publication [41] and unpublished data, topically applied Pycnogenol^® was shown to exert protective effects against photo-aging and UV damage suggesting long-lasting UV-A booster effect in solar lotions. A previous ex vivo study with human skin samples showed that a serum containing Pycnogenol^®, among other ingredients, had significant effects on air pollution-induced skin hyperpigmentation [42]. In addition, the expression of pro-inflammatory genes was reduced after application of the serum.

An in vitro study showed that Pycnogenol^® had significant inhibiting effects on skin hyperpigmentation [43]. This study showed that after incubation with Pycnogenol^®, human melanocytes that were exposed to UV-A/UV-B, near IR (IR-A) and visible light showed a significantly reduced production of melanin, endothelin-1, and PPAR (peroxisome proliferator- activated receptor) α, δ, and γ, as well as a reduction of about 66.5% of tyrosinase activity. These markers are closely associated with cutaneous pigmentation [43]. Another in vitro study showed that Pycnogenol^® protects collagen and elastin from degradation by inhibiting MMP-2 and MMP-9 (Matrix metalloproteinase-2 and 9) activity and by binding to these structural proteins [44].

This adds to previously observed effects of the topical application of Pycnogenol^®. Previously published pre-clinical studies with Pycnogenol^® have shown effects, such as wound healing [45], anti-microbial efficacy [46], and mild solar protection [41]. In an open controlled clinical study, topical application of 0.2% Pycnogenol^® for 6 weeks in individuals with acne significantly reduced porphyrin production, skin redness, and pigmentation compared to controls, showing efficacy against acne-related inflammation and discoloration [47]. These observations in addition to the results of this study make Pycnogenol^® an interesting ingredient offering a broad diversity of skin health benefits tailored for cosmetic formulations.

This study has some limitations. The experiments used skin samples from only one donor, so it does not consider natural differences between people (like age, sex, or antioxidant levels). The ex vivo skin model is good for testing topical treatments, but it doesn’t reproduce how the whole body (e.g., immune system or metabolism) would respond. This might affect how stable or effective the active compounds are in the skin. To confirm these results and better reflect real-world variability, future studies should use samples from multiple donors.

5. Conclusions

In the ex vivo human skin explant model, Pycnogenol^® dose-dependently reduced Nrf2 expression in the absence of pollutants, which may indicate a decrease in basal oxidative stress under these conditions. The extract also inhibited pollutant-induced Nrf2 over-expression and reduced AHR over-expression, while modestly increasing AHR expression in the absence of pollutants. Lipid peroxidation measured as MDA levels was significantly decreased after Pycnogenol^® application, consistent with an antioxidant effect within this experimental system.

Together with the previously reported findings with other models, these results contribute to the growing body of evidence suggesting that Pycnogenol^® exerts multi-target effects relevant to skin protection. However, as this study was conducted using ex vivo human skin explants from a single donor, the observations should be interpreted within the limitations of this model and cannot be directly extrapolated to clinical outcomes.